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目的构建和表达与肝癌细胞特异结合的双价单链抗体(bivalentsingle-chainFv,BsFv),并鉴定该双价抗体的生物活性。方法以与肝癌细胞特异结合的单链抗体(scFv)为模板,设计引物引入中间连接肽(G4S)及酶切位点AscI,通过聚合酶链反应(PCR)方法扩增出两个scFv片段,将其连入原核表达载体pAB1;构建的表达载体转化大肠埃希菌TG1,挑取单克隆细菌进行扩增,提取质粒酶切、测序鉴定。阳性菌经IPTG诱导表达,表达产物经SDS-PAGE,Westernblot鉴定,间接免疫荧光流式细胞术(FACS)测定与肝癌细胞特异结合的特性。结果经酶切鉴定以及DNA测序结果表明该BsFv构建成功,SDS-PAGE、Westernblot鉴定表明表达蛋白相对分子量与BsFv理论值一致,且表达产物同亲本scFv比较,与肝癌细胞特异结合活性增强。结论BsFv构建成功,BsFv较scFv具有明显优势,为其用于临床肿瘤的诊断和治疗奠定了基础。
Objective To construct and express bivalentsingle-chain Fv (BsFv) that specifically binds to hepatocellular carcinoma cells and to identify the biological activity of the bivalent antibody. Methods Using scFv as the template, specific primers were designed to introduce intermediate linker (G4S) and restriction enzyme site AscI. Two scFv fragments were amplified by polymerase chain reaction (PCR) The recombinant plasmid was ligated into prokaryotic expression vector pAB1. The constructed expression vector was transformed into Escherichia coli TG1. The monoclonal bacteria were picked for amplification. The plasmid was extracted and sequenced. The positive bacteria were induced by IPTG, and the expressed products were identified by SDS-PAGE and Western blot. The characteristics of specific binding to hepatoma cells were determined by indirect immunofluorescence flow cytometry (FACS). Results The results of restriction enzyme digestion and DNA sequencing showed that the constructed BsFv was successfully constructed. The SDS-PAGE and Western blot showed that the relative molecular weight of the expressed protein was consistent with the theoretical value of BsFv, and the specific binding activity of the expressed product to the hepatoma cells was enhanced compared with the parental scFv. Conclusion The successful construction of BsFv, BsFv than scFv has obvious advantages for the clinical diagnosis and treatment of tumors and laid the foundation.